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© Senckenberg Gesellschaft für Naturforschung, 2014.
177
72 (2): 177 – 192
25.7.2014
ISSN 1863-7221 (print) | eISSN 1864-8312 (online)
Unravelling the goblin spiders puzzle: rDNA phylogeny of the family Oonopidae (Araneae)
Charlotte de Busschere *, 1 , Wouter Fannes 2, Arnaud Henrard 2, 3, Eva Gaublomme 1, Rudy Jocqué 2 & Léon Baert 1
1 O.D. Taxonomy and Phylogeny, Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Charlotte de Busschere* [Charlotte. [email protected]], Eva Gaublomme [[email protected]], Léon Baert [[email protected]] — 2 Section Inver tebrates noninsects, Royal Museum for Central Africa, Tervuren, Belgium; Wouter Fannes [[email protected]], Arnaud Henrard [[email protected]], Rudy Jocqué [[email protected]] — 3 Earth and Life Institute, Biodiversity research Center, Université Catholique de Louvain, Louvain la Neuve, Belgium — * Corresponding author
Accepted 20.v.2014. Published online at www.senckenberg.de/arthropodsystematics on 18.vii.2014.
AbstractThe mega-diverse haplogyne family of goblin spiders (Oonopidae Simon, 1890) has long been among the most poorly known families of spiders. However, since the launch of the goblin spider Planetary Biodiversity Inventory project knowledge about Oonopidae is rapidly expanding. Currently, Oonopidae is placed within the superfamily Dysderoidea and is divided into three subfamilies. Nevertheless, the monophyly and internal phylogeny of this family has not yet been investigated based on DNA sequence data. Hence, this study reports the first phylogeny based on ribosomal sequence data including 37 oonopid genera and representatives of all families within the Dysderoidea. These results suggest that the majority of the oonopid genera constitute a natural group. Moreover, two subfamilies Orchestininae and Sulsulinae and several morphologically defined groups e.g. the Zyngoonops- and Dysderina-groups, were well supported. In contrast, the Pelicinus-, Stenoonops- and Scaphiella-groups were not corroborated. Although most genera represented by more than one specimen were recovered as monophyletic, our study casts doubt on the monophyly of the genus Aschnaoonops Makhan & Ezzatpanah, 2011. Further-more, the results corroborate that a low degree of body sclerotisation might be considered as a plesiomorphic trait.
Key words18S, 28S, Orsolobidae, Dysderoidea, Trogloraptoridae.
1. Introduction
Goblin spiders (Oonopidae) are small haplogyne spiders, ranging in size from 0.8 to 3 mm. They occur through-out the temperate and tropical regions of the world in a large range of habitats such as deserts, mangroves, sa-vannahs and rainforests (Jocqué & Dippenaar-Schoeman 2006; FanneS et al. 2008; GriSmaDo 2010). On a smaller scale, goblin spiders can be found in litter (ubick 2005), canopy (FanneS et al. 2008) or even in caves (harvey & eDwarD 2007). Oonopidae are also well represented in the fossil record; more precisely, several oonopid fos-
sil species have been found in various ambers (penney 2006; Dunlop et al. 2012). Most fossil species have been assigned to the genus Orchestina Simon,1882, indicating that this genus was already widespread by the end of the Cretaceous (Saupe et al. 2012). Currently goblin spiders include more than 1325 recognized species and 97 genera (platnick 2013), but this might be regarded as a fraction of the total diversity as it is estimated that between 2000 and 2500 species might exist worldwide (platnick 2006; aGnarSSon et al. 2013). This underestimation of oonopid
de Busschere et al.: Phylogeny of Oonopidae spiders
178
diversity could be related to their small size, their wide geographic distribution, their occurrence in different mi-crohabitats and the rather recent use of adequate sam-pling techniques such as knock-down fogging (platnick 2006). The U.S. National Science Foundation (NSF) funded the goblin spider Planetary Biodiversity Project (PBI) from 2006 in which more than 45 researchers were involved with the aim to revise this mega-diverse fam-ily. Since the start of this goblin spider PBI project, a tremendous amount of taxonomic work has been done with the result of a twofold increase in described species and more than 70 publications. Based upon these thor-ough revisions of several genera, a high level of morpho-logical diversity is observed within this family in traits such as carapace shape and ornamentation, arrangement and number of eyes, genital morphology and degree of body sclerotisation ranging from soft-bodied taxa e.g. Oonops Templeton, 1835 to heavily sclerotized taxa e.g. Silhouettella Benoit, 1979 (Fig. 1). Additionally, sexual dimorphism is observed in several genera e.g. Unicorn Platnick & Brescovit, 1995 and Brignolia Dumitrescu & Georgescu, 1983. However, our current knowledge of the phylogeny of the Oonopidae is severely limited and solely based upon morphological data. Hitherto, Oonopidae is placed as the sister group of the family Orsolobidae within the superfamily Dysderoidea based upon the morphological analyses of platnick et al. (1991) and ramírez (2000). The remnant families within the Dysderoidea are the Segestriidae and Dysderidae. GriSwolD et al. (2012) suggested that the recently described family Troglorap-toridae might be a primitive member of the Dysderoidea based on spinneret morphology among others. Orsolo-bidae and Oonopidae are suggested to be sister groups based upon (1) the presence of only two tarsal claws, (2) claw dentition bipectinate (although few oonopids have unipectinate claws, such as Oonops pulcher Templeton, 1835, Tapinesthis inermis Simon, 1882 or Birabenella Grismado, 2010; platnick & Dupérré 2009c; henrarD & Jocqué 2012) and (3) the presence of propriorecep-
tor bristles on the tarsi (ForSter & platnick 1985; plat-nick et al. 1991; ramírez 2000). The monophyly of the Oonopidae is based upon the presence of a completely fused, unpaired testis; female palp lacking a claw; tarsal organ flat, exposed or capsulate, with a distinctive longi-tudinal ridge originating at the proximal end of the organ; legs I and II with one more tarsal organ receptor than legs III and IV (burGer 2009; burGer & michalik 2010; platnick et al. 2012). However, monophyly of this family has not yet been adequately tested with molecular data. Additionally, the internal phylogeny of the Oonopidae is rather poorly in-vestigated. Hitherto, cladistic analyses have been limited to species-level work (henrarD & Jocqué 2012; anDria-malala & hormiGa 2013; FanneS 2013) solely based on morphological data. At the supraspecific level, platnick et al. (2012) recognized three subfamilies within the Oonopidae: the Orchestininae (containing only the soft-bodied genus Orchestina), the Sulsulinae (con taining the soft-bodied genera Sulsula Simon, 1882, Dal masula Platnick, Szüts & Ubick, 2012, Unicorn, Xiombarg Bri g noli, 1979, Puan Izquierdo, 2012 and Cor testina Knofl ach, 2009) and the Oonopinae (containing all the re maining genera, including those with a heavily scle-rotized abdomen). The Orchestininae and Sul su linae are treated as more primitive oonopid subfamilies (platnick et al. 2012) based upon the presence of a well-sclerotized sperm duct in the bulbus, a feature also found in Orso-lobidae and all other spiders (ForSter & platnick 1985; platnick et al. 2012). platnick et al. (2012) consider the soft-bodied New Zealand genus Kapitia Forster, 1956 as the sister group of other Oonopinae. Furthermore, within the higher Oonopinae, a distinction can be made be-tween soft-bodied genera such as Birabenella, Oonops, Neotrops Grismado & Ramírez, 2014 and Oonopinus Si-mon, 1893 and hard-bodied (armoured) genera (Fig. 1). These are characterized by a distinctly sclerotized abdo-men, and have been considered to constitute a mono-phyletic group (platnick & Dupérré 2010b; platnick et al. 2012). Within the armoured oonopids, six large ge-
Fig. 1. The spider family Oonopidae is characterized by a high level of morphological diversity in traits such as the degree of body sclero-tisation, ranging from heavily sclerotized taxa within the Oonopinae, e.g. Silhouettella loricatula (A), to soft-bodied oonopids, e.g. Oonops placidus (B). (Pictures from A. Henrard)
A B
179
ARTHROPOD SYSTEMATICS & PHYLOGENY — 72 (2) 2014
nus groups have so far been recognized based on puta-tive synapomorphies: the Stenoonops-group (platnick & Dupérré 2010b), the Dysderina-group (platnick & Dupérré 2011b,a, 2012), the Scaphiella-group (platnick & Dupérré 2009a, 2010c, 2011c), the Pelicinus-group (Álvarez-paDilla et al. 2012; platnick et al. 2012), the Gamasomorphoids (Álvarez-paDilla et al. 2012) and the Zyngoonops-group (FanneS 2012). However, as noted above, these subfamilies and genus groups are not based on quantitative phylogenetic analyses, and their validity thus remains to be tested. The main goals of this study are to infer the first phy-logeny of the Oonopidae based on ribosomal DNA se-quence data and to use this phylogeny to evaluate how well several morphologically defined groups delineate well supported phylogenetic relationships.
2. Materials and methods
2.1. Taxon sampling
The dataset includes 106 Oonopidae taxa representing 36 genera and stemming from different continents and several specimens representing Segestriidae, Orsolobi-dae, Dysderidae and Trogloraptoridae (Table 1). A few specimens representing the Filistatidae and Liphistiidae were included as outgroup taxa. Specimens or tissue of specimens were provided by members of the PBI pro-ject. Specimens were collected from 2001 until 2012 by different sampling techniques (mainly sieving, pitfall trapping, fogging, hand collecting). Fixation and preser-vation conditions of the collected specimens might have varied among collectors. Most specimens were sampled and stored in 70% ethanol which is common for mor-phological work. A minority of the samples was fixed and stored directly in 97% ethanol at – 20°C. Specimens were identified to family, genus and/or species level by members of the PBI project (Table 1).
2.2. DNA extraction, amplification and sequencing
Samples were extracted with a NucleoSpin® tissue kit (Macherey-Nagel, Düren) according to the manufac-turer’s protocol. Three overlapping fragments of 18S (c. 1800 nt) were amplified using the following primer pairs: 18S1F/18S5R (c. 820 nt), 18S3F/18SBI (c. 850 nt) and 18Sa2.0/18S9R (c. 650 nt) (Giribet et al. 1996). In addi-tion, partial fragments of the 28S ribosomal DNA were se-quenced with the primer pair: 28SZX1/28SC (c. 1200 nt)
(mallatt & Sullivan 1998; heDin & maDDiSon 2001). Polymerase chain reaction was initiated with an initial denaturation at 95°C for 2′30″ followed by 35 (18S, 28S) cycles. Each cycle started with a denaturation at 95°C (30″) followed by an annealing step of 30″ (annealing temperatures 18S1F/18S5R: 57°C; 18S3F/18SBI: 58°C; 18Sa2.0/18S9R: 48°C; 28SZX1/28SC: 48.5°C). Each cycle ended with an extension step at 72°C (1′30″). Gen-bank accession numbers are provided in Table 1. DNA samples are deposited in the Royal Belgian Institute of Natural Sciences (Belgium, Brussels) and stored at – 20°C. Sequence data for missing outgroup specimens were obtained from NCBI Genbank (Table 1). Approxi-mately 97% of the sequences are new from this study (Table 1).
2.3. Alignment procedures
Sequences were edited and assembled in SeqScape Soft-ware v2.5 (Applied Biosystems, Foster City, CA, USA). 18S sequences were aligned based on two methods re-sulting in (1) a MAFFT alignment, based on the E-INS-I algorithm of the program package MAFFT (katoh et al. 2002) implemented online (MAFFT version 7 (katoh & StanDley 2013), default settings), and (2) a structure informed manual alignment taking into account the es-tablished secondary structure of Androctonus australis Ewing, 1928 (Accession number: X77908 available at: http://www.rna.ccbb.utexas.edu). Based upon the infor-mation of the secondary structure of A. australis, un-paired (loop) and paired (stem) regions were delimited. 28S sequences were aligned solely based on the E-INS-I algorithm (MAFFT) as only a partial fragment of the 28S was obtained. Poorly aligned positions were eliminated from the 28S alignment by using the most relaxed op-tions in Gblocks 0.91b (caStreSana 2000; Dereeper et al. 2008). A concatenated 18S-28S alignment was created by concatenating the MAFFT 18S and 28S alignments. Estimates of average evolutionary divergence within each species, genus and family were obtained by calcu-lating the number of base differences per site within each group in MEGA5 (tamura et al. 2011).
2.4. Phylogenetic analyses
Phylogeny reconstructions were obtained using Maxi-mum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). Gaps were treated as missing data. MP analyses were conducted with PAUP*4.0b10 (SwoFForD 1993, 2002). MP analyses were initiated with 1000 random addition sequence replicates with TBR branch swapping and a time limit of 20 s per replicate in PAUP*. The most parsimonious trees were filtered from
de Busschere et al.: Phylogeny of Oonopidae spiders
180
Tabl
e 1.
Sum
mar
y of
spe
cim
en in
form
atio
n. C
olum
n “G
enus
”: u
ndes
crib
ed g
ener
a ha
ve b
een
assi
gned
five
-lette
r co
des
acco
rdin
g to
the
PBI-
conv
entio
ns. C
olum
ns “
18S”
and
“28
S”: G
enba
nk a
cces
sion
nu
mbe
rs in
ital
ics a
re n
ot fr
om th
is st
udy.
Fam
ilySu
bfam
ilyGE
NU
SSP
ECIE
SSE
XDN
A-nr
PBI-
nrCO
UN
TRY
CON
TIN
ENT
YEAR
COLL
. / D
ET.
18S
28S
Oono
pida
eOo
nopi
nae
Dysderina
tiputini
M22
015
091
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9114
KF92
9158
Oono
pida
eOo
nopi
nae
Paradysderina
fusiscuta
F26
559
7Ec
uado
rSo
uth
Amer
ica
2009
PBI e
xped
ition
/ N
. Pla
tnic
kKF
9291
15KF
9291
59
Oono
pida
eOo
nopi
nae
Paradysderina
righty
F27
258
7Ec
uado
rSo
uth
Amer
ica
2009
PBI e
xped
ition
/ N
. Pla
tnic
kKF
9291
16KF
9291
60
Oono
pida
eOo
nopi
nae
Paradysderina
vlad
M22
115
092
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9118
—
Oono
pida
eOo
nopi
nae
Paradysderina
vlad
M22
415
103
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9119
KF92
9162
Oono
pida
eOo
nopi
nae
Paradysderina
yanayacu
M20
430
842
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9117
KF92
9161
Oono
pida
eOo
nopi
nae
Scaphidysderina
cotopaxi
M26
957
4Ec
uado
rSo
uth
Amer
ica
2009
PBI e
xped
ition
/ N
. Pla
tnic
kKF
9291
20KF
9291
64
Oono
pida
eOo
nopi
nae
Scaphidysderina
tandapi
F27
855
4Ec
uado
rSo
uth
Amer
ica
2009
PBI e
xped
ition
/ N
. Pla
tnic
kKF
9291
21KF
9291
63
Oono
pida
eOo
nopi
nae
Aschnaoonops
cosanga
F20
230
839
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9106
KF92
9151
Oono
pida
eOo
nopi
nae
Aschnaoonops
silvae
M21
230
831
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9108
—
Oono
pida
eOo
nopi
nae
Aschnaoonops
tiputini
M21
130
830
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9110
KF92
9153
Oono
pida
eOo
nopi
nae
Bidysderina
perdido
M20
330
840
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9107
KF92
9152
Oono
pida
eOo
nopi
nae
Tinadysderina
otonga
F27
149
559
Ecua
dor
Sout
h Am
eric
a20
09B.
Bae
hr /
N. P
latn
ick
KF92
9109
KF92
9154
Oono
pida
eOo
nopi
nae
Tridysderina
bellavista
F26
749
544
Ecua
dor
Sout
h Am
eric
a20
09PB
I exp
editi
on /
N. P
latn
ick
KF92
9111
KF92
9155
Oono
pida
eOo
nopi
nae
Tridysderina
galeras
M26
649
536
Ecua
dor
Sout
h Am
eric
a20
09PB
I exp
editi
on /
N. P
latn
ick
KF92
9112
KF92
9157
Oono
pida
eOo
nopi
nae
Tridysderina
jatun
F20
930
834
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
—KF
9291
56
Oono
pida
eOo
nopi
nae
Tridysderina
yasuni
M22
615
105
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9113
—
Oono
pida
eOo
nopi
nae
Neoxyphinus
termitophilus
F99
4778
Arge
ntin
aSo
uth
Amer
ica
2005
M. R
amire
zKF
9291
22KF
9291
65
Oono
pida
eOo
nopi
nae
Pelicinus
sp.
M65
5981
Aust
ralia
Ocea
nia
—K.
Edw
ard
KF92
9130
KF92
9193
Oono
pida
eOo
nopi
nae
Silhouettella
loricatula
F34
4—
Fran
ceEu
rope
2012
A. H
enra
rdKF
9290
95KF
9291
75
Oono
pida
eOo
nopi
nae
Silhouettella
sp.
—25
932
76M
adag
asca
rAf
rica
2001
C. G
risw
old
KF92
9082
—
Oono
pida
eOo
nopi
nae
Escaphiella
gertschi
—28
016
983
Gala
pago
sSo
uth
Amer
ica
2009
L. B
aert
KF92
9125
—
Oono
pida
eOo
nopi
nae
Escaphiella
gertschi
—28
216
987
Gala
pago
sSo
uth
Amer
ica
2009
L. B
aert
KF92
9126
KF92
9197
Oono
pida
eOo
nopi
nae
Escaphiella
ramirezi
M13
914
892
Urug
uay
Sout
h Am
eric
a20
05M
. Ram
irez
KF92
9123
—
Oono
pida
eOo
nopi
nae
Escaphiella
schm
idti
M52
2990
2N
icar
agua
Sout
h Am
eric
a20
07C.
Viq
uez /
N. P
latn
ick
KF92
9124
KF92
9198
Oono
pida
eOo
nopi
nae
Niarchos
barragani
—27
740
4Ec
uado
rSo
uth
Amer
ica
2009
PBI e
xped
ition
/ N
. Pla
tnic
kKF
9291
29—
Oono
pida
eOo
nopi
nae
Niarchos
bonaldi
—27
4—
Ecua
dor
Sout
h Am
eric
a20
09PB
I exp
editi
on /
N. P
latn
ick
KF92
9091
—
Oono
pida
eOo
nopi
nae
Scaphios
orellana
M21
815
089
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9092
—
Oono
pida
eOo
nopi
nae
Scaphios
yanayacu
M26
842
0Ec
uado
rSo
uth
Amer
ica
2009
PBI e
xped
ition
/ N
. Pla
tnic
kKF
9290
93—
Oono
pida
eOo
nopi
nae
Birabenella
elqui
F22
814
993
Chile
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9104
—
Oono
pida
eOo
nopi
nae
Birabenella
homonota
M22
714
989
Chile
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9105
—
Oono
pida
eOo
nopi
nae
Brignolia
kapit
M29
236
337
Brun
eiAs
ia20
09C.
Gris
wol
d / Y
. Kra
nzKF
9290
56KF
9291
66
Oono
pida
eOo
nopi
nae
Brignolia
kapit
M29
336
338
Brun
eiAs
ia20
09C.
Gris
wol
d / Y
. Kra
nzKF
9290
57KF
9291
67
Oono
pida
eOo
nopi
nae
Brignolia
kapit
M29
436
317
Brun
eiAs
ia20
09C.
Gris
wol
d / Y
. Kra
nzKF
9290
58KF
9291
68
Oono
pida
eOo
nopi
nae
Cavisternum
sp.
M63
5979
Umbr
awar
raAu
stra
lia—
K. E
dwar
dKF
9290
71KF
9291
76
Oono
pida
eOo
nopi
nae
WFA
AIsp
.—
30—
Cong
oAf
rica
2006
R. J
ocqu
é / W
. Fan
nes
KF92
9083
KF92
9190
181
ARTHROPOD SYSTEMATICS & PHYLOGENY — 72 (2) 2014
Tabl
e 1
cont
inue
d.
Oono
pida
eOo
nopi
nae
WFA
AIW
FAAI
2F
113
—Gh
ana
Afric
a20
05R.
Joc
qué
/ W. F
anne
sKF
9290
85—
Oono
pida
eOo
nopi
nae
WFA
AIW
F004
—74
—Gh
ana
Afric
a20
05R.
Joc
qué
/ W. F
anne
sKF
9290
84—
Oono
pida
eOo
nopi
nae
WFA
AEsp
.F
115
—Co
ngo
Afric
a20
06R.
Joc
qué
/ W. F
anne
sKF
9290
50—
Oono
pida
eOo
nopi
nae
WFA
AJsp
.F
111
—Gu
inea
Afric
a20
08R.
Joc
qué
/ W. F
anne
sKF
9290
52—
Oono
pida
eOo
nopi
nae
WFA
AQsp
.F
50—
Cong
oAf
rica
2007
R. J
ocqu
é / W
. Fan
nes
KF92
9051
—
Oono
pida
eOo
nopi
nae
Heteroonops
spinimanus
F14
431
069
San
Fela
sco
Nor
th A
mer
ica
2007
X. W
ang
KF92
9100
KF92
9186
Oono
pida
eOo
nopi
nae
Heteroonops
spinimanus
—29
7—
Cuba
Sout
h Am
eric
a20
10N
. Pla
tnic
kKF
9291
01KF
9291
87
Oono
pida
eOo
nopi
nae
Ischnothyreus
peltifer
F28
416
986
Gala
pago
sSo
uth
Amer
ica
2009
L. B
aert
KF92
9088
KF92
9189
Oono
pida
eOo
nopi
nae
Ischnothyreus
sp.
F89
6728
Laos
Asia
2008
P. Jä
ger
KF92
9087
—
Oono
pida
eOo
nopi
nae
Ischnothyreus
sp.
F19
8—
Cong
oAf
rica
2007
R. J
ocqu
éKF
9290
86KF
9291
81
Oono
pida
eOo
nopi
nae
Ischnothyreus
sp.
M95
4777
Quee
nsla
ndAu
stra
lia20
06C.
Gris
wol
d / C
.J. G
rism
ado
KF92
9089
—
Oono
pida
eOo
nopi
nae
Lucetia
distincta
F29
8—
Cuba
Sout
h Am
eric
a20
10N
. Pla
tnic
kKF
9290
97—
Oono
pida
eOo
nopi
nae
Lucetia
sp.
F27
916
912
Gala
pago
sSo
uth
Amer
ica
2009
L. B
aert
KF92
9096
KF92
9174
Oono
pida
eOo
nopi
nae
Melchisedec
thevenot
F71
9211
Cam
eroo
nAf
rica
2007
R. J
ocqu
éKF
9291
34—
Oono
pida
eOo
nopi
nae
Melchisedec
sp.
F35
332
879
Guin
eeAf
rica
2012
A. H
enra
rdKF
9291
33—
Oono
pida
eOo
nopi
nae
Neotrops
poguazu
M13
614
910
Arge
ntin
aSo
uth
Amer
ica
2005
M. R
amire
zKF
9290
75—
Oono
pida
eOo
nopi
nae
Neotrops
labarquei
M14
014
891
Urug
uay
Sout
h Am
eric
a20
05M
. Ram
irez
KF92
9076
—
Oono
pida
eOo
nopi
nae
Neotrops
labarquei
F14
314
894
Urug
uay
Sout
h Am
eric
a20
05M
. Ram
irez
KF92
9077
KF92
9182
Oono
pida
eOo
nopi
nae
Neotrops
pombero
F94
1477
0Ar
gent
ina
Sout
h Am
eric
a20
05M
. Ram
irez
KF92
9078
KF92
9183
Oono
pida
eOo
nopi
nae
Neotrops
waorani
F20
630
836
Ecua
dor
Sout
h Am
eric
a20
05M
. Ram
irez
KF92
9079
KF92
9184
Oono
pida
eOo
nopi
nae
Neotrops
waorani
M21
330
832
Ecua
dor
Sout
h Am
eric
a20
05M
. Ram
irez
KF92
9080
KF92
9185
Oono
pida
eOo
nopi
nae
Tapinesthis
inermis
—30
9—
Belg
ium
Euro
pe20
10A.
Hen
rard
KF92
9098
KF92
9149
Oono
pida
eOo
nopi
nae
Tapinesthis
inermis
M70
—Be
lgiu
mEu
rope
2008
R. J
ocqu
éKF
9290
99KF
9291
50
Oono
pida
eOo
nopi
nae
Farqua
sp.
F11
4—
Sout
h Af
rica
Afric
a20
08R.
Joc
qué
KF92
9081
—
Oono
pida
eOo
nopi
nae
Oonops
placidus
—32
7—
Spai
nEu
rope
2010
A. H
enra
rdKF
9290
21—
Oono
pida
eOo
nopi
nae
Oonops
pulcher
F41
816
980
Fran
ceEu
rope
2012
A. H
enra
rd—
KF92
9142
Oono
pida
eOo
nopi
nae
Oonops
pulcher
F69
—Be
lgiu
mEu
rope
2008
R. J
ocqu
éKF
9290
22KF
9291
44
Oono
pida
eOo
nopi
nae
Oonops
pulcher
M42
116
977
Fran
ceEu
rope
2012
A. H
enra
rd—
KF92
9143
Oono
pida
eOo
nopi
nae
Opopaea
yukii
M14
823
366
Quee
nsla
ndAu
stra
lia20
08B.
Bae
hrKF
9290
65—
Oono
pida
eOo
nopi
nae
Opopaea
sp.
—14
923
342
Quee
nsla
ndAu
stra
lia20
08B.
Bae
hrKF
9290
63—
Oono
pida
eOo
nopi
nae
Opopaea
sp.
M36
—So
uth
Afric
aAf
rica
2008
R. J
ocqu
éKF
9290
61KF
9291
71
Oono
pida
eOo
nopi
nae
Opopaea
sp.
F49
—Ca
mer
oon
Afric
a20
07R.
Joc
qué
KF92
9059
KF92
9169
Oono
pida
eOo
nopi
nae
Opopaea
sp.
—33
—Co
ngo
Afric
a20
07R.
Joc
qué
KF92
9062
KF92
9172
Oono
pida
eOo
nopi
nae
Opopaea
deserticola
M28
516
985
Gala
pago
sSo
uth
Amer
ica
2009
L. B
aert
KF92
9060
KF92
9170
Oono
pida
eOo
nopi
nae
Opopaea
olivernashi
F14
723
284
Quee
nsla
ndAu
stra
lia20
08B.
Bae
hrKF
9290
64—
Oono
pida
eOo
nopi
nae
Opopaea
apicalis
—28
6—
Gala
pago
sSo
uth
Amer
ica
2009
L. B
aert
KF92
9066
—
Oono
pida
eOo
nopi
nae
Opopaea
sp.
M10
047
79Ar
gent
ina
Sout
h Am
eric
a20
04M
. Ram
irez
—KF
9291
73
Oono
pida
eOo
nopi
nae
Prethopalpus
tropicus
M14
610
364
Quee
nsla
ndAu
stra
lia20
04B.
Bae
hrKF
9290
67—
de Busschere et al.: Phylogeny of Oonopidae spiders
182
Tabl
e 1
cont
inue
d.
Fam
ilySu
bfam
ilyGE
NU
SSP
ECIE
SSE
XDN
A-nr
PBI-
nrCO
UN
TRY
CON
TIN
ENT
YEAR
COLL
. / D
ET.
18S
28S
Oono
pida
eOo
nopi
nae
Opopaea
sp.
M10
047
79Ar
gent
ina
Sout
h Am
eric
a20
04M
. Ram
irez
—KF
9291
73
Oono
pida
eOo
nopi
nae
Prethopalpus
tropicus
M14
610
364
Quee
nsla
ndAu
stra
lia20
04B.
Bae
hrKF
9290
67—
Oono
pida
eOo
nopi
nae
Triaeris
sp.
F14
214
886
Arge
ntin
aSo
uth
Amer
ica
2008
M. R
amire
zKF
9290
46—
Oono
pida
eOo
nopi
nae
Triaeris
sp.
F53
—Ge
rman
yEu
rope
2008
R. J
ocqu
é / W
. Fan
nes
KF92
9047
KF92
9180
Oono
pida
eOo
nopi
nae
Triaeris
sp.
F21
430
833
Ecua
dor
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9048
—
Oono
pida
eOo
nopi
nae
Triaeris
sp.
F22
914
898
Arge
ntin
aSo
uth
Amer
ica
2009
M. R
amire
zKF
9290
49KF
9291
79
Oono
pida
eOo
nopi
nae
Scaphiella
sp.
—27
6—
Ecua
dor
Sout
h Am
eric
a20
09PB
I exp
editi
on /
N. P
latn
ick
KF92
9127
KF92
9195
Oono
pida
eOo
nopi
nae
Scaphiella
sp.
M30
0—
Cuba
Sout
h Am
eric
a20
10N
. Pla
tnic
kKF
9291
28KF
9291
96
Oono
pida
eOo
nopi
nae
Oonopoides
sp.
—29
9—
Cuba
Sout
h Am
eric
a20
05N
. Pla
tnic
kKF
9291
02KF
9291
88
Oono
pida
eOo
nopi
nae
Xyphinus
sp.
—15
315
934
Thai
land
Asia
2007
P. Sc
hwen
ding
er /
Y. K
ranz
KF92
9068
—
Oono
pida
eOo
nopi
nae
Xyphinus
sp.
F29
136
321
Brun
eiAs
ia20
09Y.
Kra
nzKF
9290
70KF
9291
78
Oono
pida
eOo
nopi
nae
Xyphinus
sp.
—83
6740
Laos
Asia
2008
P. Jä
ger
KF92
9069
KF92
9177
Oono
pida
eOo
nopi
nae
Stenoonops
sp.
F30
2—
Cuba
Sout
h Am
eric
a20
10N
. Pla
tnic
kKF
9291
03—
Oono
pida
eOo
nopi
nae
Australoonops
sp.
—33
8—
Sout
h Af
rica
Afric
a20
08R.
Joc
qué
KF92
9131
—
Oono
pida
eOo
nopi
nae
Australoonops
sp.
—34
1—
Sout
h Af
rica
Afric
a20
08R.
Joc
qué
KF92
9132
—
Oono
pida
eOr
ches
tinin
aeOrchestina
sp.
F96
4774
Chile
Sout
h Am
eric
a20
05M
. Ram
irez
KF92
9053
KF92
9145
Oono
pida
eOr
ches
tinin
aeOrchestina
communis
—11
916
774
Cong
oAf
rica
2007
R. J
ocqu
éKF
9290
40—
Oono
pida
eOr
ches
tinin
aeOrchestina
communis
—15
716
775
Cong
oAf
rica
2007
R. J
ocqu
éKF
9290
41—
Oono
pida
eOr
ches
tinin
aeOrchestina
fractipes
M19
1—
Cong
oAf
rica
2009
A. H
enra
rd &
R. J
ocqu
éKF
9290
38—
Oono
pida
eOr
ches
tinin
aeOrchestina
saaristoi
F19
6—
Cong
oAf
rica
2009
A. H
enra
rd &
R. J
ocqu
éKF
9290
90KF
9291
91
Oono
pida
eOr
ches
tinin
aeOrchestina
cornuta
F18
316
798
Cam
eroo
nAf
rica
2007
R. J
ocqu
éKF
9290
44KF
9291
47
Oono
pida
eOr
ches
tinin
aeOrchestina
crypta
M18
567
74Co
ngo
Afric
a20
07R.
Joc
qué
KF92
9045
KF92
9148
Oono
pida
eOr
ches
tinin
aeOrchestina
fannesi
F15
689
14So
uth
Afric
aAf
rica
2008
R. J
ocqu
éKF
9290
72—
Oono
pida
eOr
ches
tinin
aeOrchestina
macrofoliata
M18
667
75Co
ngo
Afric
a20
07R.
Joc
qué
KF92
9039
—
Oono
pida
eOr
ches
tinin
aeOrchestina
pavesiiformis
F23
230
955
Arge
ntin
aSo
uth
Amer
ica
2010
M. R
amire
zKF
9290
94—
Oono
pida
eOr
ches
tinin
aeOrchestina
SaW
a5J
237
3091
3Ec
uado
rSo
uth
Amer
ica
2009
M. R
amire
zKF
9290
73—
Oono
pida
eOr
ches
tinin
aeOrchestina
Sum
1F
250
3087
2Ec
uado
rSo
uth
Amer
ica
2009
M. R
amire
zKF
9290
74—
Oono
pida
eOr
ches
tinin
aeOrchestina
sp.
M39
916
995
Sing
apor
eAs
ia20
12P.
Groo
taer
t / A
. Hen
rard
KF92
9042
—
Oono
pida
eOr
ches
tinin
aeOrchestina
sp.
M24
114
882
Arge
ntin
aSo
uth
Amer
ica
2008
M. R
amire
zKF
9290
55KF
9291
46
Oono
pida
eOr
ches
tinin
aeOrchestina
sp.
F13
714
909
Arge
ntin
aSo
uth
Amer
ica
2009
M. R
amire
zKF
9290
54—
Oono
pida
eOr
ches
tinin
aeOrchestina
sp.
M39
316
990
Sing
apor
eAs
ia20
12P.
Groo
taer
t / A
. Hen
rard
KF92
9043
—
Oono
pida
eSu
lsul
inae
Puan
chechehet
M41
243
348
Chile
Sout
h Am
eric
a20
10N
. Fer
retti
KF92
9023
KF92
9136
Oono
pida
eSu
lsul
inae
Unicorn
sp.
F41
542
236
Chile
Sout
h Am
eric
a20
10M
. Izq
uier
doKF
9290
24—
Oono
pida
eSu
lsul
inae
Unicorn
catleyi
M24
515
059
Arge
ntin
aSo
uth
Amer
ica
2006
M. R
amire
zKF
9290
25KF
9291
35
Orso
lobi
dae
Duripelta
borealis
—14
510
375
New
Zea
land
Ocea
nia
2005
B. B
aehr
KF92
9035
—
183
ARTHROPOD SYSTEMATICS & PHYLOGENY — 72 (2) 2014
the saved trees. Bootstrap support values were obtained with 1000 bootstrap replicates (time limit set to 60 s per bootstrap replicate). BI analyses were performed using MrBayes ver. 3.2.1 (ronquiSt & huelSenbeck 2003) available on the University of Oslo Bioportal computer cluster (kumar et al. 2009, http://www.bioportal.uio.no). In a first setup, both 18S, 28S and the 18S-28S alignments were analysed under a general time reversible (GTR; yanG 1994) model with all model parameters estimated from the data, a gamma distribution to account for site rate variation (+G) and a proportion of invariant sites (+I) as selected by jModeltest (poSaDa 2008). In the second setup, a mixed model was applied to the structural 18S alignment. The mixed model takes into account the sec-ondary structure by modelling the RNA paired (stem) re-gions separately from unpaired (loop) regions. The stem regions in which nucleotide pairing results in strong cor-relation of substitutions across sites, were modelled by the doublet model and a 4 × 4 model (GTR + I + G) was used for the loop regions. All analyses were run with two different Metropolis-coupled Markov chains (MCMC, four chains) for 10 million generations with sampling every 1000th generation. To ensure that MCMC conver-gence was reached, we evaluated the average standard deviations of split frequencies (ASDSF), the potential scale reduction factors (PSRF) and the plots of likelihood versus generation. 25% of the trees were discarded as burn-in and posterior probabilities were calculated from the remnant set of trees. ML analyses were performed us-ing the program RAxMLGUI 1.3. (SilveStro & micha-lak 2012). The GTR model with a gamma distributed rate of variation across sites was implemented for the 18S MAFFT alignment, 28S alignment and the concatenated data set. A mixed RNA/DNA model setup (RNA6A) was used to conduct the ML tree reconstruction for the 18S structural alignment. The best scoring tree (with the high-est likelihood score out of 100 heuristic searches starting from distinct randomized maximum parsimony starting trees) is visualised with bootstrap values calculated from 1000 pseudo-replicates.
3. Results
3.1. rRNA sequences
The most successful amplification results were obtained for the 18S ribosomal gene fragment. More precisely 120 taxa representing 36 oonopid genera were obtained. The amplified fragment is on average 1630 nt long and has an average GC content of 50%. 92 positions representing five loop regions were highly variable and excluded from the phylogenetic analyses. The 18S MAFFT alignment comprised 1781 positions of which 689 were variable Ta
ble
1 co
ntin
ued.
Orso
lobi
dae
Orsolobus
pucara
M41
6—
Chile
Sout
h Am
eric
a—
M. I
zqui
erdo
KF92
9026
KF92
9137
Sege
strii
dae
Ariadna
araucana
F30
4—
Chile
Sout
h Am
eric
a20
09M
. Ram
irez
KF92
9027
KF92
9140
Sege
strii
dae
Ariadna
maxima
—10
6—
Chile
Sout
h Am
eric
a20
08M
. Ram
irez
KF92
9029
KF92
9141
Sege
strii
dae
Ariadna
boesenbergi
F30
7—
Arge
ntin
aSo
uth
Amer
ica
2010
M. R
amire
zKF
9290
28—
Sege
strii
dae
Segestria
florentina
F30
5—
Arge
ntin
aSo
uth
Amer
ica
2010
M. R
amire
zKF
9290
36—
Sege
strii
dae
Segestria
sp.
——
—Sp
ain
Euro
pe—
(Bid
egar
ayB
atis
ta &
Arn
edo
2011
)JN689079
—
Dysd
erid
aeHarpactocrates
troglophilus
——
—Tu
rkey
Euro
pe—
(Bid
egar
ayB
atis
ta &
Arn
edo
2011
)JN689046
—
Dysd
erid
aeDysdera
crocata
F19
9—
Portu
gal
Euro
pe20
10A.
Hen
rard
KF92
9031
—
Dysd
erid
aeDysdera
silvatica
——
—Ca
nary
Isla
nds
Euro
pe—
(Bid
egar
ayB
atis
ta &
Arn
edo
2011
)JN689050
—
Dysd
erid
aeDysdera
erythrina
—18
0—
Belg
ium
Euro
pe—
R. J
ocqu
éKF
9290
34—
Dysd
erid
aeHarpactea
hombergi
M86
—Be
lgiu
mEu
rope
2008
R. J
ocqu
éKF
9290
33KF
9291
39
Dysd
erid
aeHarpactea
hombergi
F30
3—
Fran
ceEu
rope
2004
M. R
amire
zKF
9290
32KF
9291
38
Dysd
erid
aeParachtes
teruelis
——
—Sp
ain
Euro
pe—
(Bid
egar
ayB
atis
ta &
Arn
edo
2011
)JN689087
—
Trog
lora
ptor
idae
Trogloraptor
sp.
juv.
391
—US
AN
orth
Am
eric
a20
12P.
Jäge
rKF
9290
30KF
9291
94
Filis
tatid
aeKukulcania
hibernalis
——
——
——
(Woo
d et
al.
2012
)JX240253
—
Filis
tatid
aePritha
cf.pallida
M41
7—
Fran
ceEu
rope
2012
A. H
enra
rdKF
9290
37KF
9291
92
Liph
istii
dae
Liphistius
sp.
——
—M
alay
sia
Asia
—(H
edin
& B
ond
2006
)DQ
639767
—
de Busschere et al.: Phylogeny of Oonopidae spiders
184
(540 parsimony informative sites). The 18S structural alignment comprised 1801 positions of which 703 were variable (551 parsimony informative sites). The average level of intraspecific 18S sequence variation was 0.18% (calculated for Paradysderina vlad Platnick & Dupérré, 2011, Neotrops waorani Grismado & Ramirez, 2013, Heteroonops spinimanus Platnick & Dupérré, 2009, Or-chestina communis Henrard & Jocqué, 2012, Tapines-this inermis and Brignolia kapit Platnick et al., 2011). Intrageneric 18S sequence variation within 20 oonopid genera was on average 1.8%, which was much higher than within one dysderid genus (Dysdera Latreille, 1804: 0.5%) and the two segestriid genera (Segestria Latreille, 1804: 0.6%, Ariadna Audouin, 1826: 0.2%) (El. Suppl. Table S1). In general, 18S sequence variation across all oonopid specimens was high (average 6.4%, El. Suppl. Table S1). The 28S-rRNA dataset contained 64 taxa re-presenting 27 oonopid genera. Gblocks eliminated 13% of the 28S alignment resulting in a partial 28S fragment, which was on average 959 nt long and had an average GC content of 59%. Intraspecific (calculated for Neo-trops waorani, Tapinesthis inermis, Brignolia kapit) and intrageneric variation was on average 0.2% and 5.3% re-spectively (El. Suppl. Table S1). The 28S alignment com-prised 974 positions, of which 497 were variable (421 parsimony informative sites). The combined 18S-28S dataset contained 57 taxa (27 oonopid genera) and 2752 characters of which 1024 were variable (801 parsimony informative).
3.2. Molecular phylogenetics
Evolutionary history was reconstructed by means of BI, ML and MP carried out on the structural and MAFFT 18S alignment (Fig. 2), 28S alignment (El. Suppl. Fig. S1) and the combined 18S-28S alignment (Fig. 3). For each of these different alignments, all three analysis types (i.e. BI, ML and MP) resolved consistently similar clades of species groups within the Oonopidae, however, the de-gree of resolution varied with BI analyses tending toward more resolution and MP analyses toward less. Higher-level relationships were more difficult to resolve based on each gene separately. In contrast, different analysis types on the combined 18S-28S alignment resolved con-sistently the same higher-level relationships. One possible drawback of ribosomal DNA genes is the possibility of amplification of paralogous copies which might confound phylogenetic reconstruction (murphy et al. 2006; vink 2013; vink et al. 2011). The consistent phylogenetic results among the two rRNA-gene trees, the absence of uninterpretable sequence chromatograms, the absence of multiple PCR bands and of long-branch at-traction currently suggest no indication of the presence of paralogous copies in these datasets. Nonetheless, the possibility of paralogy should still be kept in mind and future comparison with additional genes is encouraged.
3.2.1. Oonopidae
The monophyly of the Oonopidae was consistently strongly supported by analysis of the 28S alignment (1.00 Posterior Probability (PP), 54.6 ML, 93 MP, El. Suppl. Fig. S1) and the combined 18S-28S alignment (1.00 PP, 96 ML, 70.5 MP, Fig. 3). The latter is in contrast with the less consistent results based on the 18S alignments, which contained a higher number of oonopid genera (37 versus 27). Within all analyses based on both structural and MAFFT 18S alignments, monophyly of Oonopidae was not recovered. However, BI analyses consistently produced trees with a well-supported polytomous clade containing all taxa representing Oonopidae, Dysderidae and Orsolobidae (Fig. 2). Within this clade the majority of the Oonopidae genera were recovered as a monophy-letic group but with weak support (MAFFT: 0.72 PP, Structural: 0.63 PP), Dysderidae monophyly was robustly supported (PP = 1) while in contrast the orsolobids (Orso-lobus Simon, 1893 and Duripelta Forster, 1956) were not monophyletic and varied in their phylogenetic position depending on the type of alignment and the analysis type. The latter was also observed for a small set of soft-bodied oonopid genera (Birabenella, Heteroonops and Oono-poi des) and the genus Stenoonops Simon, 1891. More precisely, the BI based on the structural 18S alignment taking into account the secondary structure reconstructs a tree in which the genera Birabenella, Heteroonops and Oono poides are monophyletic (0.97 PP) and placed as a sister clade of Orsolobus pucara Forster & Platnick, 1985 although with very weak support (0.54 PP, Fig. 2). The BI based on the MAFFT 18S alignment placed He-teroonops and Oonopoides within a well-supported clade containing the majority of the Oonopidae (0.96 PP) while Birabenella and Stenoonops were placed more as a sister clade of Duripelta borealis (tree not shown). The phylo-genetic position of Heteroonops and Oonopoides within a well-supported clade containing the majority of the Oonopidae is also recovered in the 28S phylogeny (El. Suppl. Fig. S1).
3.2.2. Intrafamilial relationships in Oonopidae
Almost all oonopid genera of which more than one specimen was included in the analyses were recovered consistently as monophyletic although five exceptions are observed, i.e. specimens of the genera Silhouettella Benoit, 1979, Opopaea Simon, 1891, Paradysderina, A schnaoonops and Unicorn. The genus Orchestina is well represented in the 18S analysis by 16 specimens sampled at different continental regions. Within this genus two well-supported clades are observed. Within the first clade two specimens sampled in Asia (Singa-pore) form a distinct group separated from specimens from Africa (Congo & Cameroon). The second clade contains specimens sampled in Africa (Congo & South Africa) and South America (Argentina, Chile & Ecua-dor). Interestingly, O. pa vesiiformis Saaristo, 2007 sam-
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Fig. 2. Phylogeny of Oonopidae and related families based on the 18S se-quence data. Majority rule consensus Bayesian tree is shown. Squares at nodes indicate per column the analy-sis method (BI: Bayesian Inference, ML: Maximum Likelihood, MP: Ma-xi mum Parsimony) and per line the type of alignment (18S structural, 18S MAFFT). Colours of squares re pre-sent bootstrap values as following: strong support (black: BI ≥ 0.95; ML, MP ≥ 0.70), moderate support (grey: 0.75 ≤ BI < 0.95; 0.60 ≤ ML < 0.70; 0.50 < MP < 0.70), weak sup-port (white: 0.50 < BI < 0.75; 0.50 < MP, ML < 0.60) and no support (square with diagonal line).
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pled in Argentina is more similar in its 18S sequence to O. fannesi Henrard & Jocqué, 2012 stemming from South Africa than to Orchestina specimens sampled in South America. The genus Opopaea is also represented by several specimens (7) stemming from different sam-pling locations. Within Opopaea, specimens from Aus-tralia (Queensland) form a well-supported group clearly differentiated from the remaining specimens sampled in Africa and the Galápagos that are, in contrast, very similar in 18S sequences. 18S sequences from different specimens of Triaeris Simon, 1891 sampled at different locations show no variation. A number of genus groups within the Oonopidae are consistently resolved across genes and analysis types (Figs. 2 – 4). Several of these genus groups are congru-ent with currently defined morphological groups. More precisely we found strong support for the monophyly of the following groups: 1) all specimens of the Dysde-rina-group, 2) the two Sulsulinae genera, 3) Opopaea and Brignolia, 4) Cavisternum Baehr, Harvey & Smith, 2010 and Xyphinus Simon, 1893 and 5) Heteroonops and Oonopoides across both genes. Based on 18S to-pologies containing most taxa, we found strong support for the monophyly of the following groups: 1) all speci-mens belonging to the Zyngoonops-group and speci-mens of Triaeris, 2) genera belonging to the Pelicinus-group, 3) Lucetia Dumitrescu & Georgescu, 1983 and Me lchisedec Fannes, 2010 and 4) a clade including Opo paea, Brignolia, Prethopalpus Baehr et al., 2012, Ca vi sternum and Xyphinus. In contrast, specimens of the Scaphiella-group were not recovered as monophyl-etic but as two distinct monophyletic groups including 1) Niarchos Platnick & Dupérré, 2010 and Scaphios Plat nick & Dupérré, 2010 and 2) Escaphiella Platnick & Dupérré, 2009 and Scaphiella Simon, 1891 respec-tively. A clear distinction between armoured versus soft-bodied Oonopidae was not recovered. However, soft-bodied Oonopidae were placed more basal within the Oono pidae.
3.2.3. Higher-level relationships
This study allows a preliminary insight in the phyloge-netic relationships among families currently placed with-in the superfamily of the Dysderoidea. Across all align-ments, monophyly of Dysderoidea sensu GriSwolD et al. (2012) is not recovered. However, we found strong sup-port for the monophyly of a group containing Oonopidae, Orsolobidae, Segestriidae and Dysderidae based on the 18S dataset (Fig. 2) and for a clade containing Oonopi-dae, Orsolobidae and Dysderidae based on the 28S and concatenated datasets (Fig. 3, El. Suppl. Fig. S1). The monophyly of Dysderidae is supported across genes and analysis types. In contrast, neither Segestriidae nor Orso-lobidae were found to be monophyletic based on the cur-rent data sets. Moreover, our phylogenies do not support a placement of Trogloraptoridae within the Dysderoidea (Figs. 2, 3, El. Suppl. Fig. S1).
4. Discussion
4.1. Monophyly and internal phylogeny of the Oonopidae
28S and combined 18S-28S data support the hypothesis that Oonopidae are monophyletic. Similarly, 18S weakly supports a clade containing the majority of the oonopid genera with exception of four genera. One of these gen-era is the soft-bodied genus Birabenella which is consist-ently placed outside the clade containing the majority of the Oonopidae genera across the type of alignment used in the BI. In contrast, the phylogenetic position of Het-eroonops, Oonopoides and Stenoonops varies across the type of alignment used in the BI. For Heteroonops and Oonopoides a similar phylogenetic position in the 28S phylogeny in comparison with the BI based on the 18S MAFFT alignment was observed supporting the position of both genera within a clade containing the majority of the Oonopidae (El. Suppl. Fig. S1). Regrettably, no 28S sequences were obtained for Birabenella and Stenoonops excluding the comparison with the 18S results. However, it is interesting to note that Birabenella’s phylogenetic position is in agreement with its plesiomorphic traits such as unipectinate claws and visible seminal ducts (GriSmaDo 2010). When Simon (1890) established the family of the Oonopidae, he classified the genera based upon the de-gree of body sclerotisation into either armoured “lori-cati”, with abdominal scuta, or soft-bodied “molles” lacking scuta (Simon 1890). Recent work states that the soft-bodied oonopids almost certainly are not monophy-letic (platnick & Dupérré 2010b; platnick et al. 2012), while in contrast, platnick et al. (2012) suggested that armoured oonopids might constitute a monophyletic group. Although both 18S and 28S topologies do not support the monophyly of these informal morphological groups, it is interesting to note that most soft-bodied gen-era included in this study were placed more basal within the Oonopidae. This phylogenetic pattern supports a low degree of body sclerotisation as a plesiomorphic trait while a higher degree of body sclerotisation seems a de-rived trait among Oonopidae. Moreover, the 28S results are in agreement with previous work that hypothesized that Orchestina and the Sulsulinae are among the most primitive oonopids (platnick et al. 2012).
4.1.1. Orchestininae
Within this study the subfamilies Sulsulinae and Orches-tininae are well supported. Interestingly, Afrotropical Orchestina species are not recovered as a monophyletic group. However, it should be noted that this is the first analysis that includes Orchestina members of different continents, something that was not tested in the phy-
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logeny of henrarD & Jocqué (2012). Within a first clade, Asian species studied here are placed together with Afro-tropical species belonging to the macrofoliata-group as defined by henrarD & Jocqué (2012). Personal observa-tions (HA) of these (undescribed) Asian species confirm the presence of modified labial setae that are characteris-tic for the macrofoliata-group (henrarD & Jocqué 2012).
Other known Asian species – e.g. Orchestina truncatula (tonG & li 2011), O. justini (SaariSto 2001) – also ap-pear to have these special setae on the labium as well as other characters diagnostic of the marcofoliata-group. Furthermore, the results remarkably support two sub-groups within the macrofoliata-group (cf. “brown clade” and “orange clade”, see henrarD & Jocqué 2012).
Fig. 3. Phylogeny of Oonopidae and related families based on the concat-enated 28S and 18S sequence data. Majority rule consensus Bayesian tree is shown. Squares at nodes indicate the analysis method (BI: Bayesian Inference, ML: Maximum Likeli-hood, MP: Maximum Parsimony). Colours of squares represent boot-strap values as following: strong sup port (black: BI ≥ 0.95; ML, MP ≥ 0.70), moderate support (grey: 0.75 ≤ BI < 0.95; 0.60 ≤ ML < 0.70; 0.50 < MP < 0.70), weak support (white: 0.50 < BI < 0.75; 0.50 < MP, ML < 0.60) and no support (square with di-agonal line).
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Although the macrofoliata-group is not supported as an exclusively African clade, the concept of the group by it-self is still well supported by particular characters that are not present in the South American members and by the current results of the molecular data. The second clade contains both South American species and Afrotropical species. These species share the presence of a posterior plate-shaped sclerite in the female genitalia (see burGer et al. 2010), a character that typifies to some species of the probosciformis-group. However, the African group in this clade, represented here only by O. fannesi and O. saaristoi (members of the “green group” in henrarD & Jocqué 2012) is perhaps the more ambiguous of all, given the low support obtained by henrarD & Jocqué (2012) and because it shares some of its own characters with many other species of the world. This idea is supported by the molecular analysis in which the South American species seem to emerge as a related group of that Afri-can clade. Interestingly, O. fannesi (known from South Africa) appears to be more similar to O. pavesiiformis (from Argentina, probably introduced from Europe, un-published data) in 18S sequence variation than to O. saaristoi. These results are in agreement with henrarD & Jocqué (2012) who concluded that the position of O. saaristoi within the clade containing O. fannesi and O. lanceolata Henrard & Jocqué, 2012 was questionable. In addition, O. fannesi and O. pavesiiformis (pers. obs. HA) share the type of male palpal tibia that is less strongly swollen than that of O. saaristoi as well as the two bris-tles emerging from a tubular projection near the anterior margin of the labium (henrarD & Jocqué 2012). The SEM illustrations of SaariSto & maruSik (2004), cor-roborated by own observations (HA), show similar struc-tures for O. pavesii that are clearly absent in O. saaristoi. In conclusion, this group of species seems to be more heterogeneous than those from macrofoliata, with some widespread characters that seem to be not exclusive of a particular fauna. In contrast, the macrofoliata-group seems to be a more homogeneous group that shares sev-eral conspicuous morphological characters restricted to some species from Africa and South Asia. However, fu-ture studies on the genus Orchestina from other regions (e.g. Europe, Australia) should test this hypothesis.
4.1.2. The Dysderina-group
The monophyly of the Dysderina-group is corroborated, and Neoxyphinus Birabén, 1953 is recovered as sister to a clade with all other taxa in the Dysderina-group. The other genera are divided in two clades, one comprising Paradysderina and Scaphidysderina Platnick & Du-pérré, 2011, the other comprising Dysderina and its al-lies (Aschnaoonops, Bidysderina Platnick et al., 2013, Tridysderina Platnick, Berniker & Bonaldo, 2013, and Tinadysderina Platnick, Berniker & Bonaldo, 2013; Figs. 2, 3). The close relationship between Paradysderina and Scaphidysderina is not surprising. These genera share an unusual instance of sexual dimorphism: in males the dor-
sal abdominal scutum is large, but in females it is greatly reduced or absent (platnick & Dupérré 2011a,b; the ge-nus Semidysderina Platnick & Dupérré, 2011, not includ-ed in this study, shows a similar sexual dimophism). In our analysis, Paradysderina is paraphyletic with respect to Scaphidysderina, suggesting that these generic names may be synonyms. However, more extensive phyloge-netic studies are needed before any firm conclusion can be drawn. In most Paradysderina and Scaphidysderina species, the females lack any trace of a dorsal scutum. However, females of P. fusiscuta Platnick & Dupérré, 2011 and S. scutata Platnick & Dupérré, 2011 appear to have a small dorsal scutum at the anterior part of the ab-domen (platnick & Dupérré 2011a,b). Interestingly, our study casts doubt on the monophyly of Aschnaoonops. This genus contains 40 species and is widely distributed in northern South America and the West Indies (platnick et al. 2013). The present analysis includes only three spe-cies of Aschnaoonops, all from Ecuador, but even this small, geographically restricted sample is not recovered as monophyletic (Fig. 2). On the basis of similarities in male genital morphology, platnick et al. (2013) have suggested that Bidysderina is more closely related to Dysderina than to Aschnaoonops. However, in the pre-sent analysis, B. perdido is closer to A. cosanga than to D. tiputini (Figs. 2, 3).
4.1.3. The Zyngoonops-, Pelicinus-, Scaphiella-, and Stenoonops-groups
The Zyngoonops-group is strongly supported in both the 18S and concatenated analyses, not surprising, given that there is considerable morphological evidence for mono-phyly (FanneS 2013). The monophyly of the Pelicinus-group is supported by the 18S data, but in the concat-enated analysis the genera Pelicinus and Silhouettella are not placed as sister taxa (Fig. 3). Our current data provide little support for the Scaphiella-group. The proposed sister relationship between Scaphiella and Escaphiella (platnick & Dupérré 2009a) is confirmed, as is the sister relationship between Niarchos and Scaphios (platnick & Dupérré 2010a), but the four genera are not recovered as a clade (Fig. 2). Future inclusion of the remaining gen-era (Pescennina and Simlops) may improve the support for the Scaphiella-group. The Stenoonops-group is re-presented in this study by Stenoonops and Australoonops which were not recovered as sister taxa (Fig. 2). platnick & Dupérré (2010b) hypothesized a close relationship be-tween these genera, but as only a few species could be examined by scanning electron microscopy, they cau-tioned that ‘this hypothesis remains poorly tested’.
4.1.4. The Opopaea-group
The genus Opopaea is one of the most species rich and widely distributed oonopid genera (platnick & Dupérré 2009b). Males of Opopaea have a greatly enlarged pal-
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pal patella that originates subbasally or medially from the femur (platnick & Dupérré 2009b; baehr 2011; bae-hr et al. 2013). Males of Prethopalpus, Malagiella and Camptoscaphiella (the latter two are not included in this study) share this feature with Opopaea. Females of Opo-paea can be morphologically confused with members of the genera Brignolia. Hence, it is not surprising that both 18S and 28S topologies recovered a strong monophyl-etic support for Opopaea and Brignolia and that a well-supported clade consisting of Brignolia, Opopaea, and Prethopalpus was recovered in the 18S phylogeny.
4.2. Dysderoidea
Our dataset allows us to obtain preliminary insights into the higher-level phylogenetic relationships within the su-perfamily of the Dysderoidea consisting of the Oonopi-dae, Orsolobidae, Segestriidae and Dysderidae. The re-cently described family Trogloraptoridae has been sug-gested to be a member of the Dysderoidea (GriSwolD et
al. 2012). Nevertheless although our results should be in-terpreted with caution given the relatively modest taxon sampling, our sequence data do not support a placement of Trogloraptoridae within the Dysderoidea as proposed by GriSwolD et al. (2012). In contrast we found good support for the grouping of the Oonopidae, Orsolobidae, Segestriidae and Dysderidae thus excluding Troglorap-toridae from this natural group.
5. Conclusion
This study reports the first sequence data and most comprehensive phylogeny including a wide array of oonopid genera in combination with representatives of the superfamily Dysderoidea. At present we suggest that the majority of the oonopid genera constitute a natural group and we highlight the need for future inclusion of remaining genera before drawing firm conclusions on the
Fig. 4. Summary phylogeny of Oo no-pidae highlighting congruence among morphological group annotations, se-quence data and inferences. Topo logy is based on majority rule Bay esian consensus tree of 18S-RNA struc-tural alignment. Squares at nodes indicate per column the data parti-tion (18S structural, 18S MAFFT, 28S, 18S-28S alignment) and per line the analysis method (BI: Bayes ian Inference, ML: Maximum Likeli-hood, MP: Maximum Parsimony). Black squares indicate that the clade was recovered in the majority rule con sensus tree of the given analy-sis, white squares indicate that the clade was not recovered and squares with diagonal line indicate that the clade was not tested due to missing se quence data for one or fewer taxa from the clade. Taxa with diamonds are classified as soft-bodied Oonopi-dae. WFAA refers to the undescribed genera belonging to the Zyngoonops-group.
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monophyly of this family. We found good support for the two subfamilies Orchestininae and Sulsulinae and several morphologically defined groups such as the Dys-derina- and Zyngoonops-groups. In contrast, the Pelici-nus-, Stenoonops- and Scaphiella-groups were not sup-ported although future inclusion of the remaining genera of these groups might improve our understanding. We suggest that a low degree of body sclerotisation might be considered as a plesiomorphic trait and corroborate that soft-bodied Oonopidae such as Orchestina and Sul-sulinae are relatively primitive oonopids. Additionally, the obtained phylogenies do not support a placement of the Trogloraptoridae within the superfamily of the Dys-deroidea.
6. Acknowledgements
This study would not have been possible without the support of U.S. National Science Foundation (grant DEB-0613754 to N. Plat-nick, R. Gillespie, G. Hormiga and P. Sierwald) and the Belgian Science Policy (Project MO/36/019 to L. Baert). Additionally, we thank the institutions, curators and collectors that have supplied specimens: A. Henrard, R. Jocqué and W. Fannes (Royal Museum of Central Africa), B. Baehr (Queensland Museum), L. Baert and P. Grootaert (Royal Belgian Institute for Natural Sciences), L. Crespo (Uni versity of Coimbra), D. Andriamalala and G. Hormiga (The George Washington University), C. Griswold (California Academy of Sciences), K. Edward (Western Australian Museum), Y. Kranz-Bal ten sperger (Natural History Museum Bern), M. Izquierdo (Mu-seo Argentino de Ciencias Naturales), N. Ferretti (Universidad Na-tional de la Plata), P. Jäger (Senckenberg), N. Platnick (American Museum of Natural History), M. Ramírez and C. Grismado (Mu-seo Argentino de Ciencias Naturales), S. Danflous (Conservatoire d’espaces naturels de Midi-Pyrénées), N. Scharff (University of Co-pen hagen). We would like to thank the two anonymous reviewers and editor for their suggestions. Ultimately, we gratefully acknow-ledge Matías Izquierdo, Norman Platnick, Cristian Grismado and Gus tavo Hormiga for their useful remarks. This paper is publication BRC 305 of the Biodiversity Research Center (Université catho li-que de Louvain). The authors have no conflict of interest to declare.
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Electronic Supplement Files
at http://www.senckenberg.de/arthropod-systematics (“Contents”)
File: debusschere&al-oonopidaephylogeny-asp2014-electronicsupplement.pdf. – Fig. S1: Phylogeny of Oonopidae and related families based on 28S sequence data. – Table S1: Estimates of average evolutionary divergence.